1. Field of the Invention
The present invention relates generally to the field of immunotherapy. More specifically, the present invention relates to using zipper peptide-modified fiber protein to target adenoviral vectors for uses in immunotherapy.
2. Description of the Related Art
The repertoire of anti-cancer strategies, which have traditionally included surgery, chemo- and radiotherapy, has recently been expanded by the employment of novel therapeutic approaches such as anti-cancer vaccination. The rationale for the development of this new treatment modality is based on convincing evidence from studies in both laboratory animals (1–4) and humans (5–7) that the immune system can recognize and destroy malignant cells. The ultimate goal of vaccination in human cancer is to achieve long-lasting, tumor-specific immunologic memory characterized by a high destructive potential and specificity, resulting in tumor eradication in the patient.
The development of anti-cancer vaccination strategies has been rationalized by the recent identification of tumor associated antigens (TAA) which may be recognized by the immune system as specific markers of cancer cells, thereby identifying these cells as the targets. These tumor associated antigens include proteins encoded by genes with mutations or rearrangements unique to tumor cells, reactivated embryonic genes, tissue-specific differentiation antigens, and a number of other self proteins (8–15). However, despite the identification of these targets, development of effective anti-cancer vaccination strategies has been limited to a large extent by the lack of means for successful vaccination against these weak, self-derived antigens. The generation of a potent anti-tumor associated antigens immune response is thus recognized as a key issue in the development of efficient anti-cancer immunization strategies.
The problem of poor immunogenicity of self-derived tumor-associated antigens can be overcome by efficient antigen presentation by dendritic cells. Current understanding of the mechanisms of immune response development suggests that efficient capture and presentation of tumor associated antigens by antigen presenting cells (APCs) is a pivotal step in eliciting strong anti-cancer immunity. In this regard, dendritic cells (DCs), so-called “professional” antigen presenting cells, play a major role in the induction of an immune response due to their ability to process and present antigen, express high levels of co-stimulatory molecules, and activate both CD4+ and CD8+ naïve T lymphocytes (16).
Dendritic cells represent a heterogeneous population of bone marrow-derived cells present at low numbers in most peripheral tissues, where they continuously sample the antigenic content of their environment by phagocytosis, macropinocytosis and receptor-mediated endocytosis. A captured antigen is then processed intracellularly, being degraded into short peptides that are loaded onto class I and class II major histocompatibility (MHC) molecules for subsequent display on the cell surface. When dendritic cells encounter local inflammatory mediators, such as tumor necrosis factor α (TNFα) or bacterial lipopolysaccharide, they become activated and undergo a series of physiologic changes leading to their terminal differentiation, a process called “dendritic cell maturation”.
Dendritic cell maturation includes redistribution of MHC molecules from intracellular endocytic compartments to the cell surface, a selective decrease of antigen and pathogen internalization activity and a marked increase in surface expression of co-stimulatory molecules for T cell activation. Maturation also entails profound changes in dendritic cell morphology, reorganization of their cytoskeleton and surface expression of several integrins and chemokine receptors that determine their migration from peripheral tissues to secondary lymphoid organs. Thus, dendritic cells serve as initiators of immune response, capturing antigen at portals of entry and delivering it in a highly immunogenic form for efficient display to T cells.
Stemming from their key function as central mediators of T cell-based immunity, the use of dendritic cells has been proposed in a number of clinical immunotherapy strategies. One of these strategies is based on the fact that immature dendritic cells present at their surface a large proportion of empty MHC class II molecules that disappear upon maturation. If these empty receptors are loaded (“pulsed”) in vitro with tumor associated antigen-specific peptides, they can then stimulate T cells. Data obtained in several animal models have demonstrated that dendritic cells pulsed with synthetic peptides corresponding to known tumor antigens or tumor-eluted peptides are capable of inducing antigen-specific cytotoxic lymphocyte (CTL) responses that lead to protection from tumor challenge and, in some instances, regression of established tumors (17, 18). The same strategy has also been tested in human clinical trials with encouraging results. Importantly, comparable cytotoxic lymphocyte activity and tumor protection have been elicited using protein-pulsed dendritic cells (16, 19, 20).
An alternative approach uses dendritic cells that are transduced with antigen-encoding cDNA or RNA rather than tumor associated antigens (TAA) themselves or tumor associated antigens-derived peptides. Such gene-modified dendritic cells offer several potential advantages over peptide- or tumor associated antigens-loaded dendritic cells. Antigenic peptides are produced by these transduced dendritic cells themselves, loaded onto and presented by MHC molecules possibly within multiple MHC alleles, and multiple and/or undefined epitopes are potentially presented. Antigenic peptides are continuously produced and loaded onto MHC molecules in transduced dendritic cells, whereas in peptide-pulsed dendritic cells only a small proportion of cell surface MHC molecules are loaded with synthetic peptide. Furthermore, cDNA encoding immuno-modulators like, for example, cytokines and chemokines can be cotransfected in addition to antigen cDNA to affect dendritic cell and T cell functions, and to modulate immune responses. Remarkably, vaccination with dendritic cells pulsed with tumor associated antigen-encoding RNA or tumor-cell-derived polyadenylated RNA can induce CTL and protective tumor immunity (21). However, traditional physical (i.e. electroporation) or chemical (e.g. cationic lipids or calcium phosphate precipitation) methods of transfection with nucleic acids have proven either ineffective or too toxic for delivery of genes into dendritic cells (16).
In order to increase the efficiency of delivery of tumor associated antigen-encoding genes to dendritic cells, natural mechanisms of virus-mediated transduction of cells have been employed. To this end, recombinant viral vectors have proved to be more efficient in delivering tumor associated antigen-encoding sequences into dendritic cells than traditional transfection methods. Retrovirus and adenovirus (Ad) vectors coding for model tumor antigens have been used to infect dendritic cells and induce both protective and therapeutic tumor immunity (2, 3, 21). However, retroviral vectors require proliferating cells for efficient infection and are characterized by a limited capacity to accommodate heterologous DNA. In addition, retroviral vectors are difficult to produce in amounts sufficient for extensive therapeutic use. In contrast, adenovirus vectors can infect both dividing and non-dividing cells, can incorporate a substantial amount of foreign DNA, and are easily propagated and purified. This set of attractive features suggests that adenoviral vectors may be a more efficient mean of dendritic cell transduction.
Several years of studies employing adenoviral vectors for transduction of dendritic cells, however, have resulted in rather controversial data on the efficiency of this method. A critical analysis of the literature reveals that in those instances where significant levels of adenoviral-mediated gene transfer to dendritic cells was reported, very high multiplicities of infection (MOIs) had to be used. For instance, Dietz et al. reported adenoviral-mediated gene transfer to human dendritic cells using an adenoviral vector only at a MOI of 5,000 virions per cell (22). Similarly, in order to achieve efficient transduction of bone marrow-derived murine dendritic cells with Ad, Kaplan et al. used an MOI of 500 infection units per cell (23), and Rea et al. transduced human dendritic cells at a MOI of 1,000 plaque forming units per cell (24). Whereas the need to use such high doses of the vector does not normally constitute a problem in “proof of concept” studies done in a laboratory, it prevents broad application of adenoviral-transduced dendritic cells as therapeutic vaccines in the clinic. Importantly, the exposure of immature dendritic cells, whose primary biological function is to capture antigen, to a high concentration of adenoviral vectors may result in the capture of adenoviral virions by the dendritic cells and elicitation of an anti-adenoviral rather than the desired anti-tumor associated antigen immune response expected from the transduction. While these considerations may not present problems with respect to ex vivo immunization of dendritic cells with adenoviral vectors, they are particularly important in the context of potential application of adenoviral-mediated transduction of dendritic cells in vivo, where high doses of adenoviral vectors administered to patients may cause severe side effects due to toxicity (25–29), thereby compromising the efficiency of the treatment. Thus, any significant improvement on adenoviral vectors' capacity to transduce dendritic cells that would allow utilization of lower viral doses with higher rates of gene transfer would be highly beneficial for the field of genetic immunization.
Recent studies designed to address the resistance of dendritic cells to adenoviral infection have revealed the molecular basis of this problem. A majority of human adenoviruses utilize a cell entry pathway that involves the primary cellular receptor, the coxsackie virus and adenovirus receptor (CAR). Expression of CAR below certain threshold levels may be a common reason for the adenoviral-refractoriness of a variety of cell targets (30). Specifically, poor efficiencies of gene transfer to dendritic cells by adenoviral vectors have been shown to correlate with low levels of CAR expression in these cells (24, 31–33). Therefore, the dependence of adenoviral-mediated transduction on the levels of CAR expressed on target dendritic cells represents a major obstacle in using adenoviral vectors for genetic immunization.
CAR-deficiency of dendritic cells and their refractoriness to adenoviral infection may be overcome by modification of adenoviral tropism to target the vector to specific receptors expressed by dendritic cells. Recent studies performed at the Gene Therapy Center at University of Alabama at Birmingham have clearly demonstrated the efficacy of this tropism modification strategy by targeting the vector to the CD40 receptor present on the surface of dendritic cells. Specifically, by employing a bispecific antibody with affinities for both the adenovirus fiber knob and CD40, a luciferase-expressing adenoviral vector was re-routed via CD40 that served the role of an alternative primary receptor for adenoviral binding. The selection of CD40 as an alternative receptor for the adenoviral vector was rationalized by the fact that this molecule, which play an important role in antigen-presentation by dendritic cells, is efficiently expressed by immature dendritic cells (16). The CD40-targeted adenoviral vector increased reporter gene expression in dendritic cells by at least two orders of magnitude as compared to untargeted Ad. Furthermore, this enhancement was blocked by ˜90% when cells were pretreated with an excess of the unconjugated anti-CD40 monoclonal antibody.
Importantly, this antibody-based targeting resulted in modulation of the immunological status of dendritic cells by inducing their maturation. This was demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a mixed lymphocyte reaction (MLR). It has been reported that activation of dendritic cells to maturity renders them resistant to the effects of dendritic cell inhibitory cytokines like IL-10 (34) as well as to direct tumor-induced apoptosis. The capacity with which murine dendritic cells can generate an immune response in vivo has been shown to correlate with the degree of their maturation (35). Moreover, based on proposals that CD40 activation may bypass CD4+ T cell help (33), a CD40-targeted adenoviral might also have applications in cases of CD4+ dysfunction. The dual role of CD40 in this schema as both a surrogate adenoviral receptor and a powerful trigger of dendritic cell maturation rationalizes further development of dendritic cell-targeting adenoviral vectors for anti-cancer immunization.
However, there is a clear need for further improvements in targeted adenoviral vectors for dendritic cell-based anti-cancer vaccination. Despite the significant advantages offered by a CD40-specific adenoviral vector targeted to dendritic cells by a bispecific antibody, the large-scale production of targeting bispecific antibody appears to be a major hurdle in the development of this technology. Not only does the production of these conjugates require the manufacture of two individual antibodies constituting the conjugate, it also necessitates efficient conjugation of the antibodies to generate a high yield of functional product. As chemical conjugation of antibodies occurs in a random manner, a significant proportion of the cross-linked antibodies loses their antigen-binding capacity. The elimination of such non-functional by-products from the conjugation reaction further complicates the entire technological scheme and decreases the yield of the desired product, thereby increasing its cost. In addition, standardization for the production of bispecific antibody is not trivial, which makes it less attractive as a means to improve Ad-based immunization of dendritic cells in the clinic. Furthermore, additional purification steps are required in order to remove from the vector preparation excessive antibodies that do not bind to adenoviral virions and may otherwise work as inhibitors of targeted gene transfer.
Thus, the prior art is deficient in methods of targeting adenoviral vectors to dendritic cells for efficient adenoviral-based immunization of dendritic cells. The methodology described in the present invention fulfills this long-standing need and desire in the art by making and using CD40-targeted adenoviral vectors containing zipper-modified fiber protein.